Nuclear hormone receptors comprise a family of ligand-dependent transcription factors that have a broad effect on gene expression, growth, and development (Aranda et al., “Nuclear Hormone Receptors and Gene Expression,” Physiol. Rev. 81:1269-1304 (2001); McKenna et al., “Nuclear Receptor Coregulators: Cellular and Molecular Biology,” Endocr. Rev. 20:321-344 (1999); McKenna et al., “Combinatorial Control of Gene Expression by Nuclear Receptors and Coregulators,” Cell 108:465-474 (2002)). These include the thyroid hormone receptors (“TRs”) for thyroid hormone (“T3”), the retinoic acid receptors (“RARs”) for all trans RA, the RARs and the retinoid X receptors (“RXRs”) for 9-cis RA, vitamin D receptor (“VDR”) for 1, 25-(OH)2 vitamin D3, glucocorticoid receptor (“GR”), progesterone receptor (“PR”), estrogen receptors (“ERs”), and peroxisome-proliferation activated receptors (“PPARs”), which are regulated by variety of lipophilic compounds. These receptors share a similar modular structure consisting of an N-terminal “A/B” domain, a DNA-binding “C” domain, and a “D, E, and F” ligand binding domain (“LBD”) (Carson-Jurica et al., “Steroid Receptor Family: Structure and Functions,” Endocr. Rev. 11:201-218 (1990); McKenna et al., “Nuclear Receptor Coregulators: Cellular and Molecular Biology,” Endocr. Rev. 20:321-344 (1999)). The LBDs of nuclear receptors are organized into twelve helical regions and the binding of ligand to the LBD of DNA bound receptor mediates a conformational change which recruits co-activators or co-regulators leading to transcriptional activation (McKenna et al., “Nuclear Receptor Coregulators: Cellular and Molecular Biology,” Endocr. Rev. 20:321-344 (1999); Toney et al., “Conformational Changes in Chicken Thyroid Hormone Receptor al Induced by Binding to Ligand or to DNA,” Biochemistry 32:2-6 (1993)).
Co-activators which have been identified include members of the p160 family (SRC-1/NCoA-1) (Kamei et al., “A CBP Integrator Complex Mediates Transcriptional Activation and AP-1 Inhibition by Nuclear Receptors,” Cell 85:403-414 (1996); Onate et al., “Sequence and Characterization of a Coactivator of the Steroid Hormone Receptor Superfamily,” Science 270:1354-1357 (1995)); TIF-2/GRIP-1/NCoA-2 (Hong et al., “GRIP1, A Novel Mouse Protein that Serves as a Transcriptional Coactivator in Yeast for the Hormone Binding Domains of Steroid Receptors,” Proc. Natl. Acad. Sci. USA 93:4948-4952 (1996); Torchia et al., “The Transcriptional Co-Activator p/CIP Binds CBP and Mediates Nuclear-Receptor Function,” Nature 387:677-684 (1997); Voegel et al., “TIF2, a 160 kDa Transcriptional Mediator for the Ligand-Dependent Activation Function AF-2 of Nuclear Receptors,” EMBO J. 15:3667-3675 (1996)); AIB1/p/CIP/ACTR/RAC3/ TRAM-1 (Anzick et al., “AIB1, A Steroid Receptor Coactivator Amplified in Breast and Ovarian Cancer,” Science 277:965-968 (1997); Chen et al., “Nuclear Receptor Coactivator ACTR is a Novel Histone Acetyltransferase and Forms a Multimeric Activation Complex with P/CAF and CBP/p300,” Cell 90:569-580 (1997); Li et al., “RAC3, A Steroid/Nuclear Receptor-Associated Coactivator that is Related to SRC-1 and TIF2,” Proc. Natl. Acad. Sci. USA 94:8479-8484 (1997); Takeshita et al., “TRAM-1, A Novel 1 60-kDa Thyroid Hormone Receptor Activator Molecule, Exhibits Distinct Properties from Steroid Receptor Coactivator-1,” J. Biol. Chem. 272:27629-27634 (1997); Torchia et al., “The Transcriptional Co-Activator p/CIP Binds CBP and Mediates Nuclear-Receptor Function,” Nature 387:677-684 (1997)), the CBP/p300 family (Chakravarti et al., “Role of CBP/P300 in Nuclear Receptor Signalling,” Nature 383:99-103 (1996); Hanstein et al., “p300 is a Component of an Estrogen Receptor Coactivator Complex,” Proc. Natl. Acad. Sci. USA 93:11540-11545 (1996); Kamei et al., “A CBP Integrator Complex Mediates Transcriptional Activation and AP-1 Inhibition by Nuclear Receptors,” Cell 85:403-414 (1996)); RIP140 (Cavailles et al., “Nuclear Factor RIP140 Modulates Transcriptional Activation by the Estrogen Receptor,” EMBO J. 14:3741-3751 (1995)); NRC/ASC-2/PRIP/RAP250/TRBP (Caira et al., “Cloning and Characterization of RAP250, A Novel Nuclear Receptor Coactivator,” J. Biol. Chem. 275:5308-5317 (2000); Ko et al., “Thyroid Hormone Receptor-Binding Protein, an LXXLL Motif-Containing Protein, Functions as a General Coactivator,” Proc. Natl. Acad. Sci. USA 97:6212-6217 (2000); Lee et al., “A Nuclear Factor, ASC-2, is a Cancer-Amplified Transcriptional Coactivator Essential for Ligand-Dependent Transactivation by Nuclear Receptors in vivo,” J. Biol. Chem. 274:34283-34293 (1999); Mahajan et al., “A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling Through CREB-Binding Protein,” Mol. Cell. Biol. 20:5048-5063 (2000); Zhu et al., “Isolation and Characterization of Peroxisome Proliferator-Activated Receptor (PPAR) Interacting Protein (PRIP) as a Coactivator for PPAR,” J. Biol. Chem. 275:13510-13516 (2000)); PGC-1 (Puigserver et al., “A Cold-Inducible Coactivator of Nuclear Receptors Linked to Adaptive Thermogenesis,” Cell 92:829-839 (1998)), ARA70 (Yeh et al., “Cloning and Characterization of a Specific Coactivator, ARA70, for the Androgen Receptor in Human Prostate Cells,” Proc. Natl. Acad. Sci. USA 93:5517-5521 (1996)); p/CAF (Blanco et al., “The Histone Acetylase PCAF is a Nuclear Receptor Coactivator,” Genes Dev. 12:1638-1651 (1998); Yang et al., “A p300/CBP-Associated Factor that Competes with the Adenoviral Oncoprotein E1A,” Nature 382:319-324 (1996)); and NRIF3, which exhibits specificity for only the TRs and the RXRs (Li et al., “NRIF3 is a Novel Coactivator Mediating Functional Specificity of Nuclear Hormone Receptors,” Mol. Cell. Biol. 19:7191-7202 (1999)). In addition to mediating effects of nuclear hormone receptors, certain co-activators also appear to enhance the activity of other transcription factors such as NF-kB, cFos, and cJun (Ko et al., “Thyroid Hormone Receptor-Binding Protein, an LXXLL Motif-Containing Protein, Functions as a General Coactivator,” Proc. Natl. Acad. Sci. USA 97:6212-6217 (2000)).
The DRIPs/TRAPs (vitamin D receptor interacting proteins/thyroid receptor-associated proteins) are another class of factors which are recruited to ligand-bound nuclear hormone receptors (e.g., VDR and TR) (Fondell et al., “Ligand Induction of a Transcriptionally Active Thyroid Hormone Receptor Coactivator Complex,” Proc. Natl. Acad. Sci. USA 93:8329-8333 (1996); Rachez et al., “Ligand-Dependent Transcription Activation by Nuclear Receptors Requires the DRIP Complex,” Nature 398:824-828 (1999)). The DRIPs and TRAPs are multi-protein complexes which appear to be similar, if not identical, and are devoid of the p160 type of co-activators. Some of the polypeptides of the DRIP/TRAP complex also appear to be a part of the SMCC, CRSP (co-factor required for promoter specificity protein (“Sp1”)) and ARC complexes (Ito et al., “Identity Between TRAP and SMCC Complexes Indicates Novel Pathways for the Function of Nuclear Receptors and Diverse Mammalian Activators,” Mol. Cell 3:361-370 (1999); Naar et al., “Composite Co-Activator ARC Mediates Chromatin-Directed Transcriptional Activation,” Nature 398:828-832 (1999); Ryu et al., “Purification of Transcription Cofactor Complex CRSP,” Proc. Natl. Acad. Sci. USA 96:7137-7142 (1999)). The DRIP/TRAP complexes associate with ligand-bound TR or VDR via a ˜220-kDa component referred to as PBP/TRAP220/DRIP205 (Fondell et al., “Ligand Induction of a Transcriptionally Active Thyroid Hormone Receptor Coactivator Complex,” Proc. Natl. Acad. Sci. USA 93:8329-8333 (1996); Rachez et al., “Ligand-Dependent Transcription Activation by Nuclear Receptors Requires the DRIP Complex,” Nature 398:824-828 (1999); Zhu et al., “Isolation and Characterization of PBP, A Protein That Interacts with Peroxisome Proliferator-Activated Receptor,” J. Biol. Chem. 272:25500-25506 (1997)) and other components of the complex interact with other transcription factors (Ito et al., “Identity Between TRAP and SMCC Complexes Indicates Novel Pathways for the Function of Nuclear Receptors and Diverse Mammalian Activators,” Mol. Cell 3:361-370 (1999); Malik et al., “The USA-Derived Transcriptional Coactivator PC2 is a Submodule of TRAP/SMCC and Acts Synergistically With Other PCs,” Mol. Cell 5:753-760 (2000); Naar et al., “Composite Co-Activator ARC Mediates Chromatin-Directed Transcriptional Activation,” Nature 398:828-832 (1999); Rachez et al., “Ligand-Dependent Transcription Activation by Nuclear Receptors Requires the DRIP Complex,” Nature 398:824-828 (1999); Ryu et al., “Purification of Transcription Cofactor Complex CRSP,” Proc. Natl. Acad. Sci. USA 96:7137-7142 (1999)).
The association of co-activators with receptors occurs through receptor-interacting LxxLL modules of the co-activator (Darimont et al., “Structure and Specificity of Nuclear Receptor-Coactivator Interactions,” Genes Dev. 12:3343-3356 (1998); Heery et al., “A Signature Motif in Transcriptional Co-Activators Mediates Binding to Nuclear Receptors,” Nature 387:733-736 (1997); Mahajan et al., “A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling Through CREB-Binding Protein,” Mol. Cell. Biol. 20:5048-5063 (2000); Mclnerney et al., “Determinants of Coactivator LXXLL Motif Specificity in Nuclear Receptor Transcriptional Activation,” Genes Dev. 12:3357-3368 (1998)), which bind to a hydrophobic cleft in the ligand-bound receptor formed by several regions of the LBD (Darimont et al., “Structure and Specificity of Nuclear Receptor-Coactivator Interactions,” Genes Dev. 12:3343-3356 (1998); Feng et al., “Hormone-Dependent Coactivator Binding to a Hydrophobic Cleft on Nuclear Receptors,” Science 280:1747-1749 (1998); Nolte et al., “Ligand Binding and Co-activator Assembly of the Peroxisome Proliferator-Activated Receptor-γ,” Nature 395:137-143 (1998)). The p160 family of co-activators, RIP140, and TRAP220/DRIP205 contain multiple LxxLL motifs (Heery et al., “A Signature Motif in Transcriptional Co-Activators Mediates Binding to Nuclear Receptors,” Nature 387:733-736 (1997)) which is consistent with the idea that a single molecule of the co-activator can bind a nuclear receptor dimer in vivo (Darimont et al., “Structure and Specificity of Nuclear Receptor-Coactivator Interactions,” Genes Dev. 12:3343-3356 (1998); McInerney et al., “Determinants of Coactivator LXXLL Motif Specificity in Nuclear Receptor Transcriptional Activation,” Genes Dev. 12:3357-3368 (1998)).
The cloning and characterization of NRC (Nuclear Receptor Co-activator) (Mahajan et al., “A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling Through CREB-Binding Protein,” Mol. Cell. Biol. 20:5048-5063 (2000)) (also referred to as ASC-2/PRIP/RAP250/TRBP) from rat and human cells which acts as a potent co-activator for nuclear hormone receptors (Mahajan et al., “A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling Through CREB-Binding Protein,” Mol. Cell. Biol. 20:5048-5063 (2000)) and other transcription factors such as cFos, cJun, and NF-kB (Ko et al., “Thyroid Hormone Receptor-Binding Protein, an LXXLL Motif-Containing Protein, Functions as a General Coactivator,” Proc. Natl. Acad. Sci. USA 97:6212-6217 (2000)) was previously reported. NRC is organized into several modular domains which appear to play an important role in its function as a co-activator/co-regulator for nuclear hormone receptors. NRC contains one functional LxxLL motif (LxxLL-1) that binds all nuclear receptors with high affinity. This appears to occur through the formation of NRC dimers, thereby contributing two LxxLL motifs to bind nuclear receptor dimers (Mahajan et al., “A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling Through CREB-Binding Protein,” Mol. Cell. Biol. 20:5048-5063 (2000)). A region containing a second LxxLL motif (LxxLL-2) appears to be highly selective for estrogen-bound ER. NRC harbors a potent N-terminal activation domain (“AD1”), which is as active as VP16 activation domain, and a second activation domain (“AD2”) which overlaps with the receptor interacting LxxLL-1 region. Receptor binding mediates a conformational change in NRC, resulting in enhanced activity of the co-activator (Mahajan et al., “A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling Through CREB-Binding Protein,” Mol. Cell. Biol. 20:5048-5063 (2000)). The C-terminal region of NRC appears to function as a modulatory domain which influences the overall activity of NRC. NRC binds CBP/p300 with high affinity in vivo (Mahajan et al., “A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling Through CREB-Binding Protein,” Mol. Cell. Biol. 20:5048-5063 (2000)) and in vitro (Ko et al., “Thyroid Hormone Receptor-Binding Protein, an LXXLL Motif-Containing Protein, Functions as a General Coactivator,” Proc. Natl. Acad. Sci. USA 97:6212-6217 (2000)) suggesting that NRC may be an important functional component of CBP/p300 complexes in the cell.
CBP and p300, which exhibit intrinsic histone acetyl transferase activity (“HAT’), function as transcriptional integrators for multiple factors including p/CAF (a HAT) (Yang et al., “A p300/CBP-Associated Factor that Competes With the Adenoviral Oncoprotein E1A,” Nature 382:319-324 (1996)), NF-kB (Perkins et al., “Regulation of NF-kappaB by Cyclin-Dependent Kinases Associated With the p300 Coactivator,” Science 275:523-527 (1997)), the STATs (Zhang et al., “Two Contact Regions Between Statl and CBP/p300 in Interferon Gamma Signaling,” Proc. Natl. Acad. Sci. USA 93:15092-15096 (1996)), nuclear hormone receptors (Chakravarti et al., “Role of CBP/P300 in Nuclear Receptor Signalling,” Nature 383:99-103 (1996); Hanstein et al., “p300 is a Component of an Estrogen Receptor Coactivator Complex,” Proc. Natl. Acad. Sci. USA 93:11540-11545 (1996); Kamei et al., “A CBP Integrator Complex Mediates Transcriptional Activation and AP-1 Inhibition by Nuclear Receptors,” Cell 85:403-414 (1996)), the p160 family (Torchia et al., “The Transcriptional Co-Activator p/CIP Binds CBP and Mediates Nuclear-Receptor Function,” Nature 387:677-684 (1997); Voegel et al., “The Coactivator TIF2 Contains Three Nuclear Receptor-Binding Motifs and Mediates Transactivation Through CBP Binding-Dependent and -Independent Pathways,” EMBO J. 17:507-519 (1998)), E1A (Chakravarti et al., “A Viral Mechanism for Inhibition of p300 and PCAF Acetyltransferase Activity,” Cell 96:393-403 (1999)), p53, (Lill et al., “Binding and Modulation of p53 by p300/CBP Coactivators,” Nature 387:823-827 (1997)), and NRC (Ko et al., “Thyroid Hormone Receptor-Binding Protein, an LXXLL Motif-Containing Protein, Functions as a General Coactivator,” Proc. Natl. Acad. Sci. USA 97:6212-6217 (2000); Mahajan et al., “A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling Through CREB-Binding Protein,” Mol. Cell. Biol. 20:5048-5063 (2000)). Although NRC appears to associate With CBP in vivo (Mahajan et al., “A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling Through CREB-Binding Protein,” Mol. Cell. Biol. 20:5048-5063 (2000)), the identity of other factors that are part of this or other NRC complexes that play a role in the action of NRC are unknown. NRC Interacting Factor-1 (“NIF-1”), which associates with and enhances the activity of NRC in vivo, is a novel nuclear protein of the recently proposed BED-finger domain family (Aravind, “The BED Finger, A Novel DNA-Binding Domain in Chromatin-Boundary-Element-Binding Proteins and Transposases,” Trends Biochem. Sci. 25:421-423 (2000)) containing six zinc-fingers which directly interacts with NRC but not with nuclear hormone receptors. Although NIF-1 does not bind directly to nuclear hormone receptors, it markedly enhances their ligand-dependent transcriptional activity in vivo. In addition, like NRC, NIF-1 also enhances the activities of cFos and cJun in vivo. Because nuclear hormone receptors are involved in human gene expression, and growth and development, the ability to regulate hormone receptors at the cellular level would provide a powerful tool for diagnosis and treatment in a wide variety of human disease conditions. What is needed now is the isolation and characterization of the nucleotide sequence of a factor which regulates nuclear hormone receptors at the molecular level. Also needed are methods using such a factor for the modulation of transcription factors in human cells, so that endocrine function and cell growth and development can be manipulated for the prevention and treatment of human disease.
The present invention is directed to overcoming these and other deficiencies in the art.